The proposed research is aimed at identifying and characterizing all functions, induced by herpes simplex virus after infection, which are involved in viral replication. Replicating viral DNA will be analyzed in infected cells with respect to its tertiary structure and mechanism of DNA strand-growth, by combining biophysical, enzymological, and electronmicroscopic techniques. The gene products ov viral temperature-sensitive DNA- mutants will be purified using complementation assays based on cell-free DNA synthesis systems. Several viral enzymes, for which no mutants are available (the alkaline DNase, the deoxypyrimidine triphosphatase, potential virus specific DNa topo-isomerase, and the latered ribonucleotide reductase), will be investigated biochemically after purification; and mutants in such "non-essential" functions will be obtained throug in vitro mutagenesis. A procedure will be developed to selectively extract proteins associated with replicating viral DNA. And, as a long term goal, the synthesis of viral enzymes from isolated viral genes will be explored. With respect to the basic biochemistry of DNA replication, the herpes simplex virus system offers the unique advantage of combining genetic and enzymological approaches in a mammalian background. With respect to herpes simplex virus induced diseases in man, the studies are expected to be of significance, because (i) the information about viral functions, which are non-essential for virus growth in cell cultures, will help to understand the mechanism of pathogenicity in animals, where such functions are likely to be essential, and (ii) because a complete catalogue of all viral replication enzymes will provide an improved basis for the analysis of the targets for antiviral chemotherapy.